Protein-Ligand Binding and Structural Modelling Studies of Pheromone-Binding Protein-like Sol g 2.1 from Solenopsis geminata Fire Ant Venom.

Sol g 2 is the major protein in Solenopsis geminata fire ant venom. It shares the highest sequence identity with Sol i 2 (S. invicta) and shares high structural homology with LmaPBP (pheromone-binding protein (PBP) from the cockroach Leucophaea maderae). We examined the specific Sol g 2 protein ligands from fire ant venom. The results revealed that the protein naturally formed complexes with hydrocarbons, including decane, undecane, dodecane, and tridecane, in aqueous venom solutions. Decane showed the highest affinity binding (Kd) with the recombinant Sol g 2.1 protein (rSol g 2.1). Surprisingly, the mixture of alkanes exhibited a higher binding affinity with the rSol g 2.1 protein compared to a single one, which is related to molecular docking simulations, revealing allosteric binding sites in the Sol g 2.1 protein model. In the trail-following bioassay, we observed that a mixture of the protein sol g 2.1 and hydrocarbons elicited S. geminata worker ants to follow trails for a longer time and distance compared to a mixture containing only hydrocarbons. This suggests that Sol g 2.1 protein may delay the evaporation of the hydrocarbons. Interestingly, the piperidine alkaloids extracted have the highest attraction to the ants. Therefore, the mixture of hydrocarbons and piperidines had a synergistic effect on the trail-following of ants when both were added to the protein.

The genome of the ant Tetramorium bicarinatum reveals a tandem organization of venom peptides genes allowing the prediction of their regulatory and evolutionary profiles.

BACKGROUND: Venoms have evolved independently over a hundred times in the animal kingdom to deter predators and/or subdue prey. Venoms are cocktails of various secreted toxins, whose origin and diversification provide an appealing system for evolutionary researchers. Previous studies of the ant venom of Tetramorium bicarinatum revealed several Myrmicitoxin (MYRTX) peptides that gathered into seven precursor families suggesting different evolutionary origins. Analysis of the T. bicarinatum genome enabling further genomic approaches was necessary to understand the processes underlying the evolution of these myrmicitoxins. RESULTS: Here, we sequenced the genome of Tetramorium bicarinatum and reported the organisation of 44 venom peptide genes (vpg). Of the eleven chromosomes that make up the genome of T. bicarinatum, four carry the vpg which are organized in tandem repeats. This organisation together with the ML evolutionary analysis of vpg sequences, is consistent with evolution by local duplication of ancestral genes for each precursor family. The structure of the vpg into two or three exons is conserved after duplication events while the promoter regions are the least conserved parts of the vpg even for genes with highly identical sequences. This suggests that enhancer sequences were not involved in duplication events, but were recruited from surrounding regions. Expression level analysis revealed that most vpg are highly expressed in venom glands, although one gene or group of genes is much more highly expressed in each family. Finally, the examination of the genomic data revealed that several genes encoding transcription factors (TFs) are highly expressed in the venom glands. The search for binding sites (BS) of these TFs in the vpg promoters revealed hot spots of GATA sites in several vpg families. CONCLUSION: In this pioneering investigation on ant venom genes, we provide a high-quality assembly genome and the annotation of venom peptide genes that we think can fosters further genomic research to understand the evolutionary history of ant venom biochemistry.

Cytotoxicity and Radiosensitizing Potentials of Pilosulin-3, a Recombinant Ant Venom, in Breast Cancer Cells.

Venom peptides are promising agents in the development of unconventional anticancer therapeutic agents. This study explored the potential of Pilosulin-3, a recombinant peptide from the venom of the Australian jack jumper ant "Myrmecia pilosula", as a cytotoxic and radiosensitizing agent in MCF-7 and MDA-MB-231 breast cancer (BC) cell lines. Pilosulin-3's cytotoxicity was evaluated across a wide range of concentrations using a proliferation assay. Cell cycle progression and apoptosis were examined at the inhibitory concentration 25% (IC25) and IC50 of Pilosulin-3, both with and without a 4Gy X-ray irradiation dose. Radiosensitivity was assessed at IC25 using the clonogenic survival assay. The study revealed that Pilosulin-3 exerted a concentration-dependent cytotoxic effect, with IC25 and IC50 values of 0.01 and 0.5 µM, respectively. In silico screening indicated high selectivity of Pilosulin-3 peptide, which was predicted to be the most likely anticancer agent (PROB = 0.997) with low hemolytic activity (PROP = 0.176). Although Pilosulin-3 exhibited a significant (p < 0.05) G2/M cell cycle arrest in combination with radiation, there was no discernible effect on apoptosis induction or cell survival following irradiation. In conclusion, Pilosulin-3 proved to be cytotoxic to BC cells and induced a cytostatic effect (G2/M arrest) when combined with radiation. However, it did not enhance the efficacy of cell killing by irradiation. While it holds potential as a cytotoxic agent in breast cancer treatment, its application as a radiosensitizer does not find support in these results.

Characterization and Localization of Sol g 2.1 Protein from Solenopsis geminata Fire Ant Venom in the Central Nervous System of Injected Crickets (Acheta domestica).

Solenopsis geminata is recognized for containing the allergenic proteins Sol g 1, 2, 3, and 4 in its venom. Remarkably, Sol g 2.1 exhibits hydrophobic binding and has a high sequence identity (83.05%) with Sol i 2 from S. invicta. Notably, Sol g 2.1 acts as a mediator, causing paralysis in crickets. Given its structural resemblance and biological function, Sol g 2.1 may play a key role in transporting hydrophobic potent compounds, which induce paralysis by releasing the compounds through the insect's nervous system. To investigate this further, we constructed and characterized the recombinant Sol g 2.1 protein (rSol g 2.1), identified with LC-MS/MS. Circular dichroism spectroscopy was performed to reveal the structural features of the rSol g 2.1 protein. Furthermore, after treating crickets with S. geminata venom, immunofluorescence and immunoblotting results revealed that the Sol g 2.1 protein primarily localizes to the neuronal cell membrane of the brain and thoracic ganglia, with distribution areas related to octopaminergic neuron cell patterns. Based on protein-protein interaction predictions, we found that the Sol g 2.1 protein can interact with octopamine receptors (OctRs) in neuronal cell membranes, potentially mediating Sol g 2.1's localization within cricket central nervous systems. Here, we suggest that Sol g 2.1 may enhance paralysis in crickets by acting as carriers of active molecules and releasing them onto target cells through pH gradients. Future research should explore the binding properties of Sol g 2.1 with ligands, considering its potential as a transporter for active molecules targeting pest nervous systems, offering innovative pest control prospects.

Discovery of an Insect Neuroactive Helix Ring Peptide from Ant Venom.

Ants are among the most abundant terrestrial invertebrate predators on Earth. To overwhelm their prey, they employ several remarkable behavioral, physiological, and biochemical innovations, including an effective paralytic venom. Ant venoms are thus cocktails of toxins finely tuned to disrupt the physiological systems of insect prey. They have received little attention yet hold great promise for the discovery of novel insecticidal molecules. To identify insect-neurotoxins from ant venoms, we screened the paralytic activity on blowflies of nine synthetic peptides previously characterized in the venom of Tetramorium bicarinatum. We selected peptide U11, a 34-amino acid peptide, for further insecticidal, structural, and pharmacological experiments. Insecticidal assays revealed that U11 is one of the most paralytic peptides ever reported from ant venoms against blowflies and is also capable of paralyzing honeybees. An NMR spectroscopy of U11 uncovered a unique scaffold, featuring a compact triangular ring helix structure stabilized by a single disulfide bond. Pharmacological assays using Drosophila S2 cells demonstrated that U11 is not cytotoxic, but suggest that it may modulate potassium conductance, which structural data seem to corroborate and will be confirmed in a future extended pharmacological investigation. The results described in this paper demonstrate that ant venom is a promising reservoir for the discovery of neuroactive insecticidal peptides.

Ant Venom-Based Ceramide Therapy Is Effective Against Atopic Dermatitis In Vivo.

BACKGROUND: Atopic dermatitis (AD) is a common skin condition with relatively few therapeutic alternatives. These include corticosteroids, which address inflammation but not superinfection, and Januse kinase (JAK) inhibitors, which have a US Food and Drug Administration (FDA) black box for potential carcinogenicity. METHODS: We demonstrate that S14, a synthetic derivative of ant venom-derived solenopsin, has potent anti inflammatory effects on the OVA murine model of atopic dermatitis. S14 has demonstrated prior activity in murine psoriasis and has the benefit of ceramide anti-inflammatory effects without being able to be metabolized into proinflammatory sphingosine-1 phosphate. RESULTS: The efficacy of S14 accompanied by the induction of IL-12 suggests a commonality in inflammatory skin disorders, and our results suggest that pharmacological ceramide restoration will be broadly effective for inflammatory skin disease. CONCLUSIONS: Solenopsin derivative S14 has anti-inflammatory effects in murine models of AD and psoriasis. This makes S14 a strong candidate for human use, and pre-IND studies are warranted.J Drugs Dermatol. 2023;22(10):1001-1006 doi:10.36849/JDD.7308.

Chemistry and Functions of Imported Fire Ant Venom.

In the United States, imported fire ants are often referred to as red imported fire ants, Solenopsis invicta Buren, black imported fire ants, S. richteri Forel, and their hybrid (S. invicta × S. richteri). Due to their aggressive stings and toxic venom, imported fire ants pose a significant threat to public health, agriculture, and ecosystem health. However, venom plays a vital role in the survival of fire ants by serving various crucial functions in defense, foraging, and colony health maintenance. Numerous reviews and book chapters have been published on fire ant venom. Due to its medical importance and the expanding global distribution of these ants, fire ant venom research remains an active and highly productive area, leading to the discovery of new components and functions. This review summarizes the recent advances in our understanding of fire ant venom chemistry and its functions within fire ant colonies.

Biological Activities and Ecological Significance of Fire Ant Venom Alkaloids.

Venoms produced by arthropods act as chemical weapons to paralyze prey or deter competitors. The utilization of venom is an essential feature in the biology and ecology of venomous arthropods. Solenopsis fire ants (Hymenoptera: Formicidae) are medically important venomous ants. They have acquired different patterns of venom use to maximize their competitive advantages rendered by the venom when facing different challenges. The major components of fire ant venom are piperidine alkaloids, which have strong insecticidal and antibiotic activities. The alkaloids protect fire ants from pathogens over the course of their lives and can be used to defend them from predators and competitors. They are also utilized by some of the fire ants' natural enemies, such as phorid flies to locate host ants. Collectively, these ants' diverse alkaloid compositions and functions have ecological significance for their survival, successful invasion, and rapid range expansion. The venom alkaloids with powerful biological activities may have played an important role in shaping the assembly of communities in both native and introduced ranges.

Fire ant Immunotherapy with inteRvals Extended to 12 weekS: The FIRES study.

BACKGROUND: There are no studies describing 12-week extended maintenance interval (EMI) immunotherapy (IT) efficacy in preventing anaphylaxis to imported fire ant (IFA) stings. OBJECTIVE: The purpose of this study was to determine the safety and efficacy of 12-week maintenance intervals in patients treated with IFA IT. METHODS: After a minimum of 3 months of conventional maintenance interval IT and verification of baseline efficacy, adults with IFA hypersensitivity were prospectively enrolled and extended their maintenance doses to 6-, 8-, and 12-week intervals. Efficacy was confirmed by means of an annual IFA sting challenge. RESULTS: A total of 25 patients initiated EMI. The severity of their initial systemic reactions was mild in 8 patients (32%), moderate in 10 patients (40%), and severe in 7 patients (28%). Maintenance IT duration at trial entry was less than 3 years in 18 patients (mean 11 months; range 3-28 months), 3 to 5 years in 4 patients (mean 46 months; range 36-57 months), and greater than 5 years in 5 patients (mean 111 months; range 67-197 months). The treatment cohort did not experience systemic reactions to extended interval injections, cluster refill injections, field stings, or sting challenges. CONCLUSION: This prospective longitudinal cohort study revealed that in adults 18 years old or older who have received at least 3 months of maintenance dose IFA-whole body extract IT with proven efficacy, extension to a 12-week EMI is a safe effective treatment option. The benefits of EMI include a reduced number of injections, clinic visits, and lapses in maintenance IT.

Envenomation From Flood-Related Fire Ant Rafting: A Cautionary Note.

BACKGROUND: Imported fire ants aggressively sting humans, leading to a variety of local and systemic effects. Fire ants display a behavioral adaptation to survive flooding, called "rafting," bringing humans into even more contact with fire ants. METHODS: To assess frequency of encounters with fire ant rafts, duck hunters on the website "Duck Hunter's Forum" were asked about their experiences with the ants while wading or boating in flooded areas. All members of the group received a brief explanation and asked to respond directly if they had had such an encounter. They were then asked to fill out a short 6-question survey. RESULTS: There were 2021 views of the thread about fire ant raft encounters, with 35 (1.7%) responses. Twenty-four (68.6%) said they had experienced encounters with fire ant rafts. Six responders described purposely or inadvertently physically touching the rafts with their body parts, boat, or a paddle, causing the ants to enter their boat or to climb directly onto the hunter(s). Five respondents were stung (number of stings from "a few" to 50), describing reactions to stings as "small pustules," "whelps," and "pimple-like, puss-filled whelps." CONCLUSIONS: These results confirm that fire ant rafts formed during flooding present a potential hazard to persons walking, wading, or boating in those areas. We suggest that these rafts are an under-appreciated health hazard. It is reasonable to assume that people in flooded areas occasionally contact these floating rafts and are stung multiple times. We provide preliminary prevention/protection recommendations for health professionals, to be shared with their patients and the general public.

Ant venoms contain vertebrate-selective pain-causing sodium channel toxins.

Stings of certain ant species (Hymenoptera: Formicidae) can cause intense, long-lasting nociception. Here we show that the major contributors to these symptoms are venom peptides that modulate the activity of voltage-gated sodium (NaV) channels, reducing their voltage threshold for activation and inhibiting channel inactivation. These peptide toxins are likely vertebrate-selective, consistent with a primarily defensive function. They emerged early in the Formicidae lineage and may have been a pivotal factor in the expansion of ants.

Antimicrobial Peptide Arsenal Predicted from the Venom Gland Transcriptome of the Tropical Trap-Jaw Ant Odontomachus chelifer.

With about 13,000 known species, ants are the most abundant venomous insects. Their venom consists of polypeptides, enzymes, alkaloids, biogenic amines, formic acid, and hydrocarbons. In this study, we investigated, using in silico techniques, the peptides composing a putative antimicrobial arsenal from the venom gland of the neotropical trap-jaw ant Odontomachus chelifer. Focusing on transcripts from the body and venom gland of this insect, it was possible to determine the gland secretome, which contained about 1022 peptides with putative signal peptides. The majority of these peptides (75.5%) were unknown, not matching any reference database, motivating us to extract functional insights via machine learning-based techniques. With several complementary methodologies, we investigated the existence of antimicrobial peptides (AMPs) in the venom gland of O. chelifer, finding 112 non-redundant candidates. Candidate AMPs were predicted to be more globular and hemolytic than the remaining peptides in the secretome. There is evidence of transcription for 97% of AMP candidates across the same ant genus, with one of them also verified as translated, thus supporting our findings. Most of these potential antimicrobial sequences (94.8%) matched transcripts from the ant's body, indicating their role not solely as venom toxins.

Testing the Novel Weapons Hypothesis of the Argentine Ant Venom on Amphibians.

The globally invasive Argentine ant (Linepithema humile) possesses a venom lethal to some amphibian species in the invaded range. To test the novel weapons hypothesis (NWH), the effects of the toxin on the cohabiting amphibian species in the ant's native range need to be investigated. The invader should benefit from the novel chemical in the invaded range, because the species are not adapted, but the venom should not be effective in the native range. We explore the venom effects on juveniles of three amphibian species with different degrees of myrmecophagy inhabiting the ant's native range: Rhinella arenarum, Odontophrynus americanus, and Boana pulchella. We exposed the amphibians to the ant venom, determined the toxic dose, and evaluated the short- (10 min to 24 h) and medium-term (14 days) effects. All amphibian species were affected by the venom independently of myrmecophagy. In addition to amphibian sensitivity, we discuss how the differential Argentine ant abundance and density in the two ranges could be the key to the susceptibility of amphibians to the venom, resulting in the possibility of NWH. Our results confirm the potential magnitude of the impact of the Argentine ant in successfully invaded areas for the conservation of already threatened amphibians.

Sensitization of Guinea Pig Skin to Imported Fire Ant Alkaloids and Establishment of an Inflammatory Model.

Imported fire ants (IFAs), Solenopsis invicta, release their venom through multiple stings that induce inflammation, allergies, shock, and even death. Although IFA venom protein sensitization and related subcutaneous immunotherapy have been studied, few studies have examined the potential toxicity or pathogenicity of alkaloids, the main substances in IFA venom. Here, IFA alkaloids were identified and analyzed by gas chromatography-mass spectrometry; we further determined an appropriate extraction method and its effectiveness for extracting high-purity alkaloids through comparative analysis and guinea pig skin sensitivity tests. The alkaloids released from the IFA abdomen included those present in the head and thorax, and the alkaloids in the abdomen accounted for the highest proportion of the total extract. The abdominal extirpation method yielded alkaloids with a purity above 97%, and the skin irritation response score and histopathological diagnosis suggest that intradermal injection of the extracted alkaloids produced symptoms effectively simulating those of IFA stings. The successful establishment of an inflammatory model in guinea pigs stung by IFAs provides a basis for further research on the mechanism of inflammatory diseases caused by IFAs.

Proteinaceous Venom Expression of the Yellow Meadow Ant, Lasius flavus (Hymenoptera: Formicidae).

Ants are one of the important groups of venomous animals with about 14,000 described species. Studies so far focused on the discovery of venom proteins are only available for limited stinging ants, and the proteinaceous compositions of the stingless ants are completely unknown. Here, we used the transcriptomic approach to identify venom components from the yellow meadow ant, Lasius flavus, a stingless ant. The transcriptomic analysis yielded an extraordinary simplicity of the venom expression profile, with 17 venom proteins, such as phospholipase B, odorant binding protein, and apolipoprotein D. Ten of them were discovered as novel toxins for future functional investigations. Quantitative real time PCR analysis revealed that genes encoding the identified venom proteins display exclusively or highly expression profiles in venom glands, validating them as venom compositions. Our findings contribute to the understanding of the evolutional diversity of toxins between stinging and stingless ants.

Spread of stinging ants to oceanic islands, and the need to raise awareness of prevention and treatment of ant stings.

OBJECTIVE: Venomous invasive ants are rapidly dispersing throughout oceanic islands. Medics unfamiliar with envenomation or venom-induced anaphylaxis may be unprepared for the range of possible reactions and corresponding treatments. We detail the suboptimal treatment of a patient suffering anaphylaxis from an ant sting on a remote island and describe what treatment should have been provided. METHODS: The patient experienced stings on his feet from an ant later identified as tropical fire ant, Solenopsis geminata. Clinical examination revealed throat swelling without obstruction of the airway or pharynx. RESULTS: The patient was provided the following suboptimal treatment: intravenously-administered antihistamine and saline perfusion. Injected epinephrine should be the standard first line of treatment for anaphylaxis, even when not all symptoms are present. CONCLUSION: A rise in invasive hymenopteran stings on oceanic islands is inevitable, and proactively improving public awareness and medical training could save lives.

Transcriptomic and biochemical analysis from the venom gland of the neotropical ant Odontomachus chelifer.

The genus Odontomachus is widely distributed in neotropical areas throughout Central and South America. It is a stinging ant that subdues its prey (insects) by injecting them a cocktail of toxic molecules (venom). Ant venoms are generally composed of formic acid, alkaloids, hydrocarbons, amines, peptides, and proteins. Odontomachus chelifer is an ant that inhabits neotropical regions from Mexico to Argentina. Unlike the venom of other animals such as scorpions, spiders and snakes, this ant venom has seldom been analyzed comprehensively, and their compositions are not yet completely known. In the present study, we performed a partial investigation of enzymatic and functional activities of O. chelifer ant venom, and we provide a global insight on the transcripts expressed in the venom gland to better understand their properties. The crude venom showed phospholipase A2 and antiparasitic activities. RNA sequencing (Illumina platform) of the venom gland of O. chelifer generated 61, 422, 898 reads and de novo assembly Trinity generated 50,220 contigs. BUSCO analysis against Arthropoda_db10 showed that 92.89% of the BUSCO groups have complete gene representation (single-copy or duplicated), while 4.05% are only partially recovered, and 3.06% are missing. The 30 most expressed genes in O. chelifer venom gland transcriptome included important transcripts involved in venom function such as U-poneritoxin (01)-Om1a-like (pilosulin), chitinase 2, venom allergen 3, chymotrypsin 1 and 2 and glutathione S-transferase. Analysis of the molecular function revealed that the largest number of transcripts were related to catalytic activity, including phospholipases. These data emphasize the potential of O. chelifer venom for prospection of molecules with biotechnological application.